Comparison the Effect of Bromelain Enzyme, Phosphoric Acid, and Polyacrylic Acid Treatment on Microleakage of Composite and Glass Ionomer Restorations

Statement of the Problem:
 Resin modified glass-ionomer cement (RMGIC) shows low microleakage values. Bromelain enzyme is a deproteinizing agent with an anti-inflammatory effect in human body.Efective cavity treatment is an important factor in reduction of microleakage. 
Purpose:
 The aim of this study was to determine the effectiveness of the deproteinizing aspect of 10% bromelain enzyme on the microleakage of RMGIC and composite restorations. 
Materials and Method:
 In this experimental study, 40 non-carious extracted human molar teeth were categorized in eight experimental groups (n=5). Standard class V cavities were prepared on the buccal and lingual surfaces of the teeth (n=10). The specimen were classified as Group 1, in which 20% polyacrylic acid (PAA) was applied on the teeth then treated with 10% bromelain enzyme; Group 2: 10% bromelain enzyme was applied; Group 3: 10% bromelain enzyme was applied and then treated with polyacrylic acid; Group 4: 20% polyacrylic acid was applied. Groups1 to 4 were restored with RMGIC (Fuji II LC, GC, Japan). Group 5: etched by 37% phosphoric acid and then treated by 10% bromelain; Group 6: 10% bromelain enzyme was applied without etching; Group 7: teeth were deproteinized with 10% bromelain enzyme and then etched with 37% phosphoric acid; and Group 8: cavities were etched with 37% phosphoric acid. In the groups 5 to 8, Adper single bond (3M, ESPE, USA) and filled with composite resin Z350 (3M, ESPE, USA). After thermocycling, the teeth were sectioned. Microleakage scores were measured using stereomicroscope (40×). Kruskal-Wallis and Mann-Whitney tests were used for data analysis. (p< 0.05) 
Results:
 Statistical analysis did not show any significant difference in occlusal and gingival margin microleakage in glass ionomer groups (1-4) (occlusal p= 0.218, gingival p= 0.192). Kruskal-Wallis revealed significant difference in occlusal and gingival margin microleakage of Groups 5 to 8 (occlusal p= 0.006 and gingival p= 0.00). Group 5 demonstrated the lowest occlusal microleakage (occlusal mean=0.00). 
Conclusion:
 Applying bromelain or polyacrylic acid did not affect the microleakage of glass ionomer filling. Due to the antinflamatory effects of bromelain, we suggest using it instead of PAA. Pretreatment of 10% bromelain enzyme after phosphoric acid significantly decreased microleakage in the occlusal and gingival margin of composite filling.


Introduction
Request for aesthetic restoration has led to the introduct-ion of different tooth-colored restorative materials such as glass ionomer cements (GICs) and composite resins.
GICs adhere to the enamel and dentin with fluoride release, and have low cytotoxicity and microleakage [1][2][3]; however, they have low toughness and strength [4][5][6][7]. Composite resin has an important role in esthetic dentistry, but polymerization and shrinkage cause a volumetric reduction of resin. Rapid polymerization and volume loss may lead to gap formation and debonding that cause breakdown in the margine of the restoration.
Conditioning is required for achieving an effective adhesion between the tooth structure and the GICs. Polyacrylic acid (PAA) is a traditional conditioner in GICs restorations. Conditioning of the dentin causes the chemical reaction between GIC and hydroxyapatite crystals and can demineralize partially the dentine surface [8].
The microleakage causes the permeability of chemical ions and bacteria and leads to postoperative sensitivity, recurrent caries, pulpal pathology, and failure of the restoration. Many strategies have been used to increase restoration bond strength, reduce the microleakage, and future failures such as degradation of collagen fiber [9][10][11][12]. One of these materials is bromelain, which is a proteolytic enzyme extracted from pineapple. It has many properties like reducing tissue inflammation, pain, and edema [13]. Investigation revealed that application of bromelain enzyme leads to the removal of collagen network and a significant decrease in the global leakage of the adhesive system [14]. Moreover, it has been reported that removal of unsupported dentin collagen fibers with bromelain enzyme after acid etching results acceptable bond strength [15].
As there were a few investigations that report the effect of bromelain on microleakage of composite and RMGIC restorations, this study was conducted to determine the exact function of bromelain enzyme and PAA and phosphoric acid treatment of cavity in composite and RMGIC restorations.

Materials and Method
A total of 40 human intact extracted third molars were selected in this experimental study. They were stored in 0.1% thymol solution for 48h. They were mounted 4 mm apically to CEJ in cylindrical acrylic resin with 6 cm height and 3 cm diameter.
Diamond fissure bur (330, SS White, USA) was used for every 5 preparations in a high-speed handpiece with water and air spray. Class V cavities (3mm in width, 5mm in length and 2mm in depth) [17] on the buccal and lingual surfaces of each tooth was prepared, where the gingival margin of cavities was 1 mm below the CEJ. The teeth were randomly placed into 8 groups of 10 cavities and then filled as follows.
In the Group 1, 20% PAA (GC, Tokyo, Japan) was applied for 20 s by a microbrush, rinsed for 20 s with water, and dried gently. Bromelain powder (Salamat Parmoon Amin manufacture, Iran) was dissolved in distilled water to obtain 10% bromelain enzyme. The solution was applied on the cavity surfaces by a microbrush for 60 s, rinsed for 20s and dried [12].
In the Group 2,the bromelain enzyme was applied directly into the cavities for 60 s, rinsed for 20 s, and s. In the group 7, the cavities were treated by bromelain enzyme for 60 s, rinsed for 20 s, and then 37% phosphoric acid gel was applied for 20 s on all part of cavity according to manufacture recommendation [10]. The teeth were rinsed and gently air dried and Adper single bond was applied and cured.
In the group 8, 37% phosphoric acid gel was applied for 20 s, rinsed for 20 s, and gently dried. Then Adper

Results
The means and standard deviations (SD) of microleakage scores are illustrated in Tables 1-4 and Figure 2.

Means of Kruskal-Wallis test in occlusal margin
showed that group 5 (Phosphoric acid+ Bromelain) had the lowest score (mean=0.00) compared to composite and RMGIC.

Discussion
Treatment of dentine surface with phosphoric acid leads to dissolving mineral component of the smear layer and remaining amorphous protein layer, which decreases the rate of adhesive resin penetration and forms a weak hybrid layer that subsequently reduces the composite bond strength [10]. Bromelain enzyme can eliminate organic component and collagen from the surface of the dentin and increase resin penetration into dentin structure, improve the hybrid layer, and decrease microleakage [20].
In our study, pretreatment of 10% bromelain en- Therefore, in the current study, bromelain was applied for one minute and then was washed.
In one study [16], the effect of phosphoric acidetched dentin surface with 5% bromelain enzyme and Nd:YAG laser prior to the use of etch and rinse adhesive systems on microleakage margins of class V composite restorations has been reported. They showed that gingival margins microleakages were significantly higher than the occlusal margins. Thus, they concluded that application of proteolytic agents on acid-etched dentin surface prior to the application of adhesive has no significant effect on marginal microleakage of class V composite restorations [16]. This finding is opposed to our results. The difference may be related to the priority and arrangement of our study, in which bromelain was applied after phosphoric acid, and also the bromelain dilution amount, laser application, and the type of composite used. Moreover, in our study, application of bromelain after acid etch in the occlusal margin showed better results; however, in both occlusal and gingival margin, it caused a significant reduction in microleakage [23].
In our study, the statistical analysis showed that etching dentin, with 37% phosphoric acid alone, did not decrease the microleakage significantly.
In one study [24], researchers examined the etching effects of phosphoric acid versus a combination of phosphoric and hydrofluoric acid by evaluation of microleakage in a composite restoration bonded with different dentin adhesive systems. This study showed that a combination of phosphoric and hydrofluoric acid led to significant reduction in dye penetration compared to phosphoric acid conditioning per se [24]. This result suggests that phosphoric acid would not be able to decrease microleakage when used alone. In our study, when phosphoric acid was applied alone, most microleakages were observed in the gingival margin and a considerable leakage was seen in the occlusal margin. It seems the combination of phosphoric acid with another material such as hydrofluoric acid or bromelain enzyme can increase the capability of phosphoric acid to reduce microleakage.
The use of conditioning before placing the GICs sig-nificantly increases the ionic bond to dentin. PAA is a very weak acid that does not significantly demineralize dentinal tissue nor increase the likelihood of postoperative sensitivity [25]. It has been reported that pretreatment with a weak PAA conditioner has the ability to remove the smear layer and partially demineralize the dentin [26]. In our study, we applied PAA before GIC and it could reduce the microleakage to some extent.
However, the difference was not significantly different when bromelain was used alone and even in the gingival margin, application of bromelain enzyme alone showed better results than occlusal margin concerning the microleakages.  [29]. In their study, CHX was kept in the cavity but in our study, bromelain was rinsed. Therefore, bromelain enzyme application for more than 60s in the cavity may show better results. The results of their study are similar to ours but the mechanism of action differs prominently between bromelain and CHX.
In one research [30], the microleakage of RMGIC be explained by the ability of bromelain enzyme to remove the collagen network from acid-etched dentin efficiently. As a result, an increase may occur and the diffusion potential of the monomer to the intact dentin and the microleakage would be minimized. Furthermore, removing the collagen network from acid-etched dentin substrate will make the chemical composition of dentin more similar to that of enamel by minimizing the organic component of dentin substrate and this will lead to the changing of the hydrophilic properties of the dentin [11]. Future in vivo studies should be done because it differs from in vitro considering the absence of saliva, different microorganism colonization, pH, thermal condition, nutrition, and variation of tooth morphology.
In addition, we suggest conducting similar studies using different types of bonding systems with variable pH, self-etchants, and different concentration of bromelain.

Conclusion
Considering the limitations of this research, using bromelain alone in RMGIC fillings showed almost a similar microleakage in occlusal and gingival margin of PAA group. The most important result of this research is that bromelain can be used as an organic and harmless material with less damaging effect on tissues instead of chemical hazardous acid in operative dentistry. In this study, from composite filling, we concluded that the application of bromelain after phosphoric acid reduces microleakage in occlusal and gingival margins. Hence, it is recommended to use bromelain in composite fillings.